Allogeneic MSCs being used in current human clinical trials are nonclonal, i.e., such MSCs may have other types of cells in the final stem cell product. Some concerns exist about the heterogeneity of these nonclonal MSCs that are isolated and expanded by the conventional density-gradient centrifugation method. Recently, we developed a new protocol, called the subfractionation culturing method (SCM), to generate single-cell-derived clonal MSC (cMSC) lines from whole bone marrow aspirate without employing any centrifugation step for mononuclear cells and enzyme treatment process. This method allowed us to rapidly establish single-cell-derived human clonal MSC (hcMSC) lines from raw bone marrow aspirates and to establish a library of these hcMSC lines (Song et al., 2008).